DNA polymerases synthesize formation of DNA molecules that are complementary to all or a portion of a nucleic acid template. Upon hybridization of a primer to the single-stranded template, polymerases synthesize DNA in the 5′ to 3′ direction, i.e., successively adding nucleotides to the 3′-hydroxyl group of the growing strand. Thus, for example, in the presence of deoxynucleoside triphosphates (dNTPs) and a primer, a new DNA molecule, complementary to the single stranded nucleic acid template, can be synthesized. Typically an RNA or DNA template is used for synthesizing a complementary DNA molecule. However, other templates, such as chimeric templates or modified nucleic acid templates are also usable for synthesizing complementary molecules of polymerized nucleic acids. A DNA-dependent DNA polymerase utilizes a DNA template and produces a DNA molecule complementary to at least a portion of the template. An RNA-dependent DNA polymerase, i.e. a reverse transcriptase, utilizes an RNA template to produce a DNA strand complementary to at least a portion of the template, i.e., a cDNA. A common application of reverse transcriptase has been to transcribe mRNA into cDNA. Some DNA polymerases have both DNA-dependent DNA polymerase activity and RNA-dependent DNA polymerase activity.
In addition to a polymerase activity, DNA polymerases may possess one or more additional catalytic activities. Typically, DNA polymerases may have a 3′-5′ exonuclease (“proofreading”) and a 5′-3′ exonuclease activity. Each of these activities has been localized to a particular region or domain of the protein. For example, when E. coli polymerase I (pol I) is cleaved into two fragments by subtilisin, the larger (“Klenow”) fragment has 3′-5′ exonuclease and DNA polymerase activities and the smaller fragment has 5′-3′ exonuclease activity.
DNA polymerases have been isolated from a variety of mesophilic and thermophilic organisms. DNA polymerases from thermophilic organisms typically have a higher optimum temperature for polymerization activity than enzymes isolated from mesophilic organisms. Thermostable DNA polymerases have been discovered in a number of thermophilic bacterial species, including, but not limited to, Thermus aquaticus (Taq), Thermus filiformis (Tfi), Thermus thermophilus (Tth), and species of the Bacillus, Thermococcus, Sulfolobus and Pyrococcus genera. In addition, thermostable DNA polymerases from a variety of other thermophiles are described in PCT WO 03/025132, the entire contents of which are incorporated herein by reference. Thermostable DNA polymerases have been exploited in numerous applications, including the polymerase chain reaction (PCR).
PCR is used to amplify a target nucleic acid by denaturation of the target DNA, hybridization of oligonucleotide primers to specific sequences on opposite strands of the target DNA molecule, and subsequent extension of these primers with a DNA polymerase, usually a thermostable DNA polymerase, to generate two new strands of DNA which then serve as templates for a further round of hybridization and extension. If the polymerase is thermostable, then there is no need to add fresh polymerase after every denaturation step since heat will not have destroyed the polymerase activity. In RT-PCR, a DNA primer is hybridized to a strand of the target RNA molecule, and subsequent extension of this primer with a reverse transcriptase generates a new strand of DNA (i.e., cDNA), which can serve as a template for PCR.
Thermostable DNA polymerases from Thermus aquaticus (Taq) made PCR feasible. Other thermostable polymerases having different properties (e.g., higher or lower fidelity; additional, enhanced, fewer or reduced catalytic activities; altered substrate use or preference; or different cofactor requirements) suitable for particular applications have been isolated from other organisms and/or made using recombinant DNA techniques.